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WO2005080375A1 - Cristalline d'un compose biphenyle - Google Patents

Cristalline d'un compose biphenyle Download PDF

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Publication number
WO2005080375A1
WO2005080375A1 PCT/US2005/004392 US2005004392W WO2005080375A1 WO 2005080375 A1 WO2005080375 A1 WO 2005080375A1 US 2005004392 W US2005004392 W US 2005004392W WO 2005080375 A1 WO2005080375 A1 WO 2005080375A1
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Prior art keywords
salt
hydroxy
crystalline
ester
biphenyl
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Inventor
Robert Chao
Miroslav Rapta
Pierre-Jean Colson
Junning Lee
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Innoviva Inc
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Theravance Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the present invention relates to a novel crystalline naphthalene- 1 ,5-disulfonic acid salts of a biphenyl compound which are expected to be useful for treating pulmonary disorders.
  • This invention also relates to pharmaceutical compositions comprising such crystalline biphenyl compounds or prepared from such crystalline biphenyl compounds, processes and intermediates for preparing such crystalline biphenyl compounds and methods of using such crystalline biphenyl compounds to treat a pulmonary disorder.
  • the compound, biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8- hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester is specifically disclosed in this application as possessing both muscarinic antagonist and ⁇ 2 adrenergic receptor agonist activity.
  • Therapeutic agents useful for treating pulmonary disorders are advantageously administered directly into the respiratory tract by inhalation, hi this regard, several types of pharmaceutical inhalation devices have been developed for administering therapeutic agents by inhalation including dry powder inhalers (DPI), metered-dose inhalers (MDI) and nebulizer inhalers.
  • DPI dry powder inhalers
  • MDI metered-dose inhalers
  • nebulizer inhalers When preparing pharmaceutical compositions and formulations for use in such devices, it is highly desirable to have a crystalline form of the therapeutic agent that is neither hygroscopic nor deliquescent and which has a relatively high melting point (i.e. greater than about 150 °C) thereby allowing the material to be micronized without significant decomposition.
  • No salt forms of the compound for formula I have been reported previously. Accordingly, a need exists for a stable, non-deliquescent salt form of the compound of formula I which has an acceptable level of hygroscopicity and a relatively high melting point.
  • the present invention provides a crystalline naphthalene- 1, 5 -disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro- quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester or a solvate thereof.
  • a crystalline naphthalene- 1,5-disulfonic acid salt of the compound of formula I has been found not to be deliquescent, even when exposed to the atmosphere moisture.
  • a crystalline salt of this invention has been found to have an acceptable level of hygroscopicity and a very high melting point greater than 200 °C.
  • a crystalline naphthalene- 1,5-disulfonic acid salt of the compound of formula I is expected to be useful for preparing pharmaceutical compositions for treating pulmonary disorders.
  • the present invention provides a pharmaceutical composition prepared from a crystalline naphthalene- 1,5-disulfonic acid salt of the compound of formula I, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and a naphthalene-l,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)- 2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4- yl ester or a solvate thereof, h a particular embodiment, the pharmaceutical composition of this invention further comprises a steroidal anti-inflammatory agent, such as a corticosteroid; or a phosphodiesterase-4 inhibitor.
  • a steroidal anti-inflammatory agent such as a corticosteroid
  • a phosphodiesterase-4 inhibitor such as a corticosteroid
  • the compound of formula I has both muscarinic antagonist and ⁇ adrenergic receptor agonist activity. Accordingly, the naphalene- 1,5-disulfonic acid salt of this invention is expected to be useful for treating pulmonary disorders, such as asthma and chronic obstructive pulmonary disease.
  • this invention is directed to a method for treating a pulmonary disorder, the method comprising administering to a patient in need of treatment a therapeutically effective amount of provides a pharmaceutical composition prepared from a crystalline naphthalene- 1,5-disulfonic acid salt of the compound of formula I, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and a naphthalene- 1,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid 1 - ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo- 1 ,2-dihydro-quinolin-5- yl)ethylamino]nonyl ⁇ piperidin-4-yl ester or a solvate thereof.
  • this invention is directed to a method of producing bronchodilation in a patient, the method comprising administering to a patient a bronchodilation-producing amount of a provides a pharmaceutical composition prepared from a crystalline naphthalene- 1,5-disulfonic acid salt of the compound of formula I, the pharmaceutical composition comprising a pharmaceutically acceptable carrieriand a naphthalene-l,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)- 2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4- yl ester or a solvate thereof.
  • This invention is also directed to a method of treating chronic obstructive pulmonary disease or asthma, the method comprising admimstering to a patient in need of treatment a therapeutically effective amount of a provides a pharmaceutical composition prepared from a crystalline naphthalene- 1,5-disulfonic acid salt of the compound of formula I, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and a naphthalene- 1,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)- 2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4- yl ester or a solvate thereof.
  • this invention is also directed to processes for preparing a crystalline naphthalene- 1,5-disulfonic acid salt of the compound of formula I. Accordingly, in another of its method aspects, this invention provides a process for preparing a crystalline naphthalene- 1,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy- 2-oxo-l ,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester or a solvate thereof; the process comprising contacting biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2- hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester with 1,5-naphthalenedisulfonic acid.
  • this invention provides a process for preparing crystalline naphthalene- 1,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid 1- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo- 1 ,2-dihydro-quinolin-5- yl)ethylamino]nonyl ⁇ piperidin-4-yl ester or a solvate thereof, the process comprising: (a) contacting a compound of formula II:
  • step (b) contacting the product from step (b) with naphthalene-l,5-disulfonic acid to form a crystalline naphthalene-l,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid 1- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ - ⁇ iperidin-4-yl ester or a solvate thereof, wherein step (a) and (b) are conducted in the same reaction vessel without isolation of the product of step (a).
  • this invention is directed to a process for purifying biphenyl-2- ylcarbamic acid 1 - ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo- 1 ,2-dihydro-quinolin-5- yl)ethylamino]nonyl ⁇ piperidin-4-yl ester; the process comprising forming a crystalline naphthalene- 1,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy- 2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester.
  • This invention is also directed to the products prepared by the processes described herein.
  • This invention is also directed to a crystalline naphthalene-l,5-disulfonic acid salt ofbiphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro- quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester or a solvate thereof for use in therapy or as a medicament.
  • this invention is directed to the use of a crystalline naphthalene- 1,5- disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2- oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester or a solvate thereof for the manufacture of a medicament; especially for the manufacture of a medicament for the treatment of a pulmonary disorder.
  • This invention is also directed to a crystalline naphthalene- 1,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro- quinolin-5-yl)ethylamino]nonyl ⁇ iperidin-4-yl ester or a solvate thereof, in micronized form; and to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a crystalline naphthalene-l,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5- yl)ethylamino]nonyl ⁇ piperidin-4-yl ester or a solvate thereof, in micronized form.
  • Figure 1 shows an x-ray powder diffraction (XRPD) pattern of a crystalline naphthalene-l,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy- 2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester of this invention.
  • XRPD x-ray powder diffraction
  • Figure 2 shows a differential scanning calorimetry (DSC) trace and a thermal gravimetric analysis (TGA) trace for a crystalline naphthalene-l,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro- quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester of this invention.
  • DSC differential scanning calorimetry
  • TGA thermal gravimetric analysis
  • Figure 3 shows a dynamic moisture sorption (DMS) trace for a crystalline naphthalene- 1,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy- 2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester of this invention.
  • DMS dynamic moisture sorption
  • Figure 4 shows an infrared (IR) absorption spectra for a crystalline naphthalene- 1,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy- 2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester of this invention.
  • IR infrared
  • Figure 5 is a micrographic image of a crystalline naphthalene-l,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro- quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester of this invention.
  • This invention provides a crystalline naphthalene- 1,5-disulfonic acid salts of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro- quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester or a solvate thereof.
  • the active agent in these salts i.e., the compound of formula I
  • solvate means a complex or aggregate formed by one or more molecules of a solute, i.e. a naphthalene- 1,5-disulfonic acid salt of the compound of formula I, and one or more molecules of a solvent. Such solvates typically have a substantially fixed molar ratio of solute and solvent.
  • Representative solvents include, by way of example, water, methanol, ethanol, isopropanol, acetic acid and the like. When the solvent is water, the solvate formed is a hydrate.
  • therapeutically effective amount means an amount sufficient to effect treatment when administered to a patient in need of treatment.
  • treating means the treating or treatment of a disease or medical condition (such as COPD) in a patient, such as a mammal (particularly a human) that includes: (a) preventing the disease or medical condition from occurring, i.e., prophylactic treatment of a patient; (b) ameliorating the disease or medical condition, i.e., eliminating or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, i.e., slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating the symptoms of the disease or medical condition in a patient.
  • a disease or medical condition such as COPD
  • a mammal particularly a human
  • unit dosage form refers to a physically discrete unit suitable for dosing a patient, i.e., each unit containing a predetermined quantity of the salt of the invention calculated to produce the desired therapeutic effect either alone or in combination with one or more additional units.
  • unit dosage forms maybe capsules, tablets, pills, and the like.
  • a crystalline naphthalene-l,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid 1- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo- 1 ,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ - piperidin-4-yl ester of this invention can be prepared from bi ⁇ henyl-2-ylcarbamic acid 1- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ - piperidin-4-yl ester and naphthalene- 1,5-disulfonic acid.
  • naphthalene-l,5-disulfonic acid salts of this invention typically contains between about 0.75 and about 1.25 molar equivalents of naphthalene-l,5-disulfonic acid per molar equivalent of the compound of formula I; including between about 0.90 and about 1.05 molar equivalents of naphthalene- 1,5-disulfonic acid per molar equivalent of the compound of formula I.
  • the molar ratio of naphthalene- 1,5-disulfonic acid to biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ - piperidin-4-yl ester can be readily determined by various methods available to those skilled in the art. For example, such molar ratios can be readily determined by 1 H NMR. Alternatively, elemental analysis and HPLC methods can be used to determine the molar ratio.
  • biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2- dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester employed in this invention can be readily prepared from commercially available starting materials and reagents using the procedures described in the Examples below; or using the procedures described in the commonly-assigned U.S. application described in the Background section of this application.
  • Naphthalene- 1,5-disulfonic acid also known as Armstrong's Acid
  • the naphthalene-l,5-disulfonic acid employed in this invention is essentially anhydrous.
  • the biphenyl-2-ylcarbamic acid 1- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ - piperidin-4-yl ester is typically contacted with about 0.75 to about 1.25 molar equivalents of naphthalene-l,5-disulfonic acid.
  • this reaction is conducted in an inert diluent at a temperature ranging from about 0 °C to about 40 °C; including about 20 °C to about 35 °C, such as about 25 °C to about 30 °C.
  • Suitable inert diluents for this reaction include, but are not limited to, methanol, ethanol, isopropanol, isobutanol, ethyl acetate and the like.
  • a particular inert diluent is methanol.
  • the inert diluent have a low water content i.e., the inert diluent is essentially anhydrous.
  • the crystalline naphthalene- 1,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro- quinolin-5-yl)ethylamino]nonyl ⁇ -piperidin-4-yl ester is isolated from the reaction mixture by any conventional means, such as precipitation, concentration, centrifugation and the like.
  • a crystalline naphthalene-l,5-disulfonic acid salt of a compound of formula I can be prepared by contacting a silyl-protected derivative of the compound of formula I (i.e., a compound of formula II) with a source of fluoride ion and then, in the same reaction vessel, contacting the product with naphthalene- 1,5-disulfonic acid.
  • the silyl-protecting group is a tert-butyldimethylsilyl group.
  • silyl-protecting groups include tert-butyldiphenylsilyl, diphenylmethylsilyl, di- tert-buylmethylsilyl, tert-butoxydiphenylsilyl and the like.
  • the source of fluoride ion used in this process can be any reagent containing or comprising fluoride ion or hydrogen fluoride. In a particular embodiment, the source of fluoride ion is tetrabutylammonimn fluoride.
  • Other suitable sources of fluoride ion include, triethylamine trihydrofluoride, potassium fluoride with 18-crown-6, hydrogen fluoride, pyridine hydrofluoride, and the like.
  • this process is conducted in an inert diluent at a temperature ranging from about 0 °C to about 50 °C; including about 25 °C to about 45 °C, such as about 35 °C to about 40 °C.
  • Suitable inert diluents for this reaction include, but are not limited to, THF, dichloromethane, methanol and mixtures thereof.
  • a solution of biphenyl-2-yl-carbamic acid l- ⁇ 9-[(R)-2-(tert-butyldimethylsilanyloxy)-2-(8- hydroxy-2-oxo-l,2-dihydroquinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester is contacted with about 1.8 to about 3.0 molar equivalents of tetrabutylammonium fluoride in THF at about 40 °C for about 5 to 12 hours or until removal of the silyl group is substantially complete.
  • a crystalline naphthalene- 1,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2- dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester is isolated from the reaction mixture by any conventional means, such as precipitation, concentration, centrifugation and the like.
  • any conventional means such as precipitation, concentration, centrifugation and the like.
  • the crystalline naphthalene-l,5-disulfonic acid salts of this invention have a purity greater than 95%; and typically greater than 98%, as determined by high performance liquid chromatography.
  • a crystalline naphthalene-l,5-disulfonic acid salt of the present invention is characterized by an x-ray powder diffraction (XRD) pattern having two or more diffraction peaks at 2 ⁇ values selected from 11.95 ⁇ 0.25, 12.88 ⁇ 0.25, 13.88 ⁇ 0.25, 18.43 ⁇ 0.25, 20.45 ⁇ 0.25, 22.98 ⁇ 0.25, 23.83 ⁇ 0.25, and 25.94 ⁇ 0.25.
  • XRD x-ray powder diffraction
  • a crystalline form is characterized by an x-ray powder diffraction pattern comprising diffraction peaks at 2 ⁇ values of 18.43 ⁇ 0.25, 20.45 ⁇ 0.25, and 23.83 ⁇ 0.25.
  • x-ray powder diffraction pattern comprising diffraction peaks at 2 ⁇ values of 18.43 ⁇ 0.25, 20.45 ⁇ 0.25, and 23.83 ⁇ 0.25.
  • relative peak heights of XRPD spectra are dependent on a number of factors relating to sample preparation and instrument geometry, while peak positions are relatively insensitive to experimental details.
  • a crystalline naphthalene-l,5-disulfonic acid salt of the compound of formula I is characterized by an x-ray powder diffraction pattern in which the peak positions are substantially in accordance with those shown in Figure 1.
  • a crystalline naphthalene- 1,5-disulfonic acid salt of the compound of formula I is characterized by its infrared (IR) absorption spectrum which shows significant absorption bands at 689 ⁇ 1, 706 ⁇ 1, 749 ⁇ 1,768 ⁇ 1, 806 ⁇ 1, 841 ⁇ 1, 994 ⁇ 1, 1065 ⁇ 1, 1098 ⁇ 1, 1165 ⁇ 1, 1221 ⁇ 1, 1252 ⁇ 1, 1283 ⁇ 1,1437 ⁇ 1, 1474 ⁇ 1, 1533 ⁇ 1, 1613 ⁇ 1, and 1668 ⁇ 1 cm "1 , as illustrated in Figure 4.
  • IR infrared
  • a crystalline naphthalene-1 ,5-disulfonic acid salt of the compound of formula I is characterized by its differential scanning calorimetry (DSC) trace which shows an onset of endothermic heat flow at about 200 °C, as illustrated in Figure 2.
  • DSC differential scanning calorimetry
  • a crystalline naphthalene- 1,5-disulfonic acid salt of the compound of formula I has been demonstrated to have a reversible sorption/desorption profile with an acceptable, moderate level of hygroscopicity (i.e., less than 5% weight gain when exposed to atmospheric moisture). Additionally, a crystalline naphthalene- 1,5-disulfonic acid salt of the compound of formula I has been found to be stable upon exposure to elevated temperature and humidity. For example, after storage for 28 days at 40 °C and 75 % relative humidity, analysis by high pressure liquid chromatography (HPLC) showed no observable chemical degradation (i.e., less than 0.5% degradation).
  • compositions and Formulations The naphthalene- 1,5-disulfonic acid salts of the compound of formula I are typically administered to a patient in the form of a pharmaceutical composition or formulation.
  • Such pharmaceutical compositions may be administered to the patient by any acceptable route of administration including, but not limited to, inhaled, oral, nasal, topical (including transdermal) and parenteral modes of administration.
  • routes of administration including, but not limited to, inhaled, oral, nasal, topical (including transdermal) and parenteral modes of administration.
  • the salt may be dissolved in a suitable carrier.
  • this invention is directed to a pharmaceutical composition prepared from a crystalline salt of the invention, the composition comprising a pharmaceutically acceptable carrier or excipient and a naphthalene- 1,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy- 2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester or a solvate thereof.
  • such pharmaceutical compositions may contain other therapeutic and/or formulating agents if desired.
  • compositions of this invention typically contain a therapeutically effective amount of a naphthalene- 1,5-disulfonic acid salt of biphenyl-2- ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5- yl)ethylamino]nonyl ⁇ piperidin-4-yl ester or a solvate thereof.
  • such pharmaceutical compositions will contain from about 0.01 to about 95% by weight of the active agent; including, from about 0.01 to about 30% by weight; such as from about 0.01 to about 10% by weight of the active agent. Any conventional carrier or excipient may be used in the pharmaceutical compositions of this invention.
  • a particular carrier or excipient, or combinations of carriers or exipients will depend on the mode of administration being used to treat a particular patient or type of medical condition or disease state.
  • the preparation of a suitable pharmaceutical composition for a particular mode of administration is well within the scope of those skilled in the pharmaceutical arts.
  • ingredients for such compositions are commercially available from, for example, Sigma, P.O. Box 14508, St. Louis, MO 63178.
  • conventional formulation techniques are described in Remington: The Science and Practice of Pharmacy, 20 th Edition, Lippincott Williams & White, Baltimore, Maryland (2000); and H.C. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th Edition, Lippincott Williams & White, Baltimore, Maryland (1999).
  • compositions which can serve as pharmaceutically acceptable carriers include, but are not limited to, the following: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium magnesium
  • compositions of this invention are typically prepared by throughly and intimately mixing or blending a salt of the invention with a pharmaceutically acceptable carrier and one or more optional ingredients. If necessary or desired, the resulting uniformly blended mixture can then be shaped or loaded into tablets, capsules, pills, canisters, cartridges, dispensers and the like using conventional procedures and equipment. h one embodiment, the pharmaceutical compositions of this invention are suitable for inhaled administration. Suitable pharmaceutical compositions for inhaled administration will typically be in the form of an aerosol or a powder. Such compositions are generally administered using well-known delivery devices, such as a nebulizer inhaler, a metered-dose inhaler (MDI), a dry powder inhaler (DPI) or a similar delivery device.
  • MDI metered-dose inhaler
  • DPI dry powder inhaler
  • the pharmaceutical composition comprising the active agent is administered by inhalation using a nebulizer inhaler.
  • a nebulizer inhaler typically produce a stream of high velocity air that causes the pharmaceutical composition comprising the active agent to spray as a mist that is carried into the patient's respiratory tract.
  • the active agent when formulated for use in a nebulizer inhaler, is typically dissolved in a suitable carrier to form a solution.
  • the active agent can be micronized and combined with a suitable carrier to form a suspension of micronized particles of respirable size, where micronized is typically defined as having about 90% or more of the particles with a diameter of less than about 10 ⁇ m.
  • Suitable nebulizer devices are provided commercially, for example, by PARI GmbH (Starnberg, German). Other nebulizer devices include Respimat (Boehringer higelheim) and those disclosed, for example, in U.S. Patent No. 6,123,068 and WO 97/12687.
  • a representative pharmaceutical composition for use in a nebulizer inhaler comprises an aqueous solution comprising from about 0.05 ⁇ g/mL to about 10 mg/mL of a naphthalene- 1,5-disulfonic acid salt of compound of formula I or a solvate thereof.
  • the aqueous nebulizer formulation is isotonic.
  • the pharmaceutical composition comprising the active agent is administered by inhalation using a dry powder inhaler.
  • dry powder inhalers typically administer the active agent as a free-flowing powder that is dispersed in a patient's air-stream during inspiration.
  • the active agent is typically formulated with a suitable excipient such as lactose or starch.
  • the active agent is micronized and combined with a suitable carrier to form a suspension of micronized particles of respirable size, where "micronized particles” or “micromzed form” means at least about 90% of the particles have a diameter of less than about 10 ⁇ m.
  • a representative pharmaceutical composition for use in a dry powder inhaler comprises dry lactose having a particle size between about 1 ⁇ m and about 100 ⁇ m and micronized particles of a naphthalene-l,5-disulfonic acid salt of compound of formula I, or a solvate thereof.
  • a dry powder formulation can be made, for example, by combining the lactose with the active agent and then dry blending the components.
  • the active agent can be formulated without an excipient.
  • the pharmaceutical composition is then typically loaded into a dry powder dispenser, or into inhalation cartridges or capsules for use with a dry powder delivery device.
  • Examples of dry powder inhaler delivery devices include Diskhaler (GlaxoSmithKline, Research Triangle Park, NC) (see, e.g., U.S. Patent No. 5,035,237); Diskus (GlaxoSmithKline) (see, e.g., U.S. Patent No. 6,378,519; Turbuhaler (AstraZeneca, Wilmington, DE) (see, e.g., U.S. Patent No. 4,524,769); Rotahaler (GlaxoSmithKline) (see, e.g., U.S. Patent No. 4,353,365) and Handihaler (Boehringer higelheim). Further examples of suitable DPI devices are described in U.S. Patent Nos.
  • the pharmaceutical composition comprising the active agent is administered by inhalation using a metered- dose inhaler.
  • metered-dose inhalers typically discharge a measured amount of the active agent or a pharmaceutically acceptable salt thereof using compressed propellant gas.
  • pharmaceutical compositions administered using a metered-dose inhaler typically comprise a solution or suspension of the active agent in a liquefied propellant.
  • Any suitable liquefied propellant may be employed including chlorofluorocarbons, such as CC1 3 F, and hydro fluoroalkanes (HFAs), such as 1,1,1,2- tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-he ⁇ tafluoro-n- ⁇ ro ⁇ ane, (HFA 227).
  • chlorofluorocarbons such as CC1 3 F
  • HFAs hydro fluoroalkanes
  • HFA 134a 1,1,1,2- tetrafluoroethane
  • HFA 227 1,1,1,2,3,3,3-he ⁇ tafluoro-n- ⁇ ro ⁇ ane
  • co-solvents such as ethanol or pentane
  • surfactants such as sorbitan trioleate, oleic acid, lecithin, and glycerin.
  • a representative pharmaceutical composition for use in a metered-dose inhaler comprises from about 0.01 % to about 5 % by weight of a naphthalene- 1,5-disulfonic acid salt of compound of formula I, or a solvate thereof; from about 0 % to about 20 % by weight ethanol; and from about 0 % to about 5 % by weight surfactant; with the remainder being an HFA propellant.
  • Such compositions are typically prepared by adding chilled or pressurized hydrofluoroalkane to a suitable container containing the active agent, ethanol (if present) and the surfactant (if present).
  • a suspension formulation can be prepared by spray drying a coating of surfactant on micronized particles of the active agent. See, for example, WO 99/53901 and WO 00/61108.
  • WO 99/53901 and WO 00/61108 For additional examples of processes of preparing respirable particles, and formulations and devices suitable for inhalation dosing see U.S. Patent Nos.
  • compositions of this invention are suitable for oral administration.
  • suitable pharmaceutical compositions for oral administration may be in the form of capsules, tablets, pills, lozenges, cachets, dragees, powders, granules; or as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup; and the like; each containing a predetermined amount of a salt of the present invention as an active ingredient.
  • compositions of this invention When intended for oral administration in a solid dosage form (i.e., as capsules, tablets, pills and the like), the pharmaceutical compositions of this invention will typically comprise a salt of the present invention as the active ingredient and one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate.
  • such solid dosage forms may also comprise: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar- agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and/or sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and/or glycerol monostearate; (8) absorbents, such as kaolin and/or bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid poly
  • antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodium sulfite and the like; (2) oil- soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylencdiamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodium sulfite and the like
  • oil- soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (
  • Coating agents for tablets, capsules, pills and like include those used for enteric coatings, such as cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PNAP), hydroxypropyl methylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymers, cellulose acetate trimellitate (CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methyl cellulose acetate succinate (HPMCAS), and the like.
  • enteric coatings such as cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PNAP), hydroxypropyl methylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymers, cellulose acetate trimellitate (CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methyl cellulose acetate succinate (HPMCAS), and the like.
  • the pharmaceutical compositions of the present invention may also be formulated to provide slow or controlled release of the active ingredient using, byway of example, hydroxypropyl methyl cellulose in varying proportions; or other polymer matrices, liposomes and/or microspheres.
  • the pharmaceutical compositions of the present invention may optionally contain opacifying agents and may be formulated so that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Suitable liquid dosage forms for oral administration include, by way of illustration, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • Such liquid dosage forms typically comprise the active ingredient and an inert diluent, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (esp., cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • an inert diluent such as, for example, water or other solvents, solubilizing agents and emulsifier
  • Suspensions in addition to the active ingredient, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • the pharmaceutical compositions of this invention are preferably packaged in a unit dosage form.
  • such unit dosage forms may be capsules, tablets, pills, and the like.
  • the salts of this invention can also be administered transdermally using known transdermal delivery systems and excipents.
  • a compound of this invention can be admixed with permeation enhancers, such as propylene glycol, polyethylene glycolm monolaurate, azacycloalkan-2-ones and the like, and incorporated into a patch or similar delivery system. Additional excipients including gelling agents, emulsifiers and buffers, maybe used in such transdermal compositions if desired.
  • the pharmaceutical compositions of this invention may also contain other therapeutic agents that are co-administered with a naphthalene-l,5-disulfonic acid salt of compound of formula I, or solvate thereof.
  • the pharmacuetical compositions of this invention may further comprise one or more therapeutic agents selected from other ⁇ 2 adrenergic receptor agonists, anti-inflammatory agents (e.g. steroidal anti-inflammatory agents, such as corticosteroids; and non-steroidal anti- inflammatory agents (NSATDs), phosphodiesterase IN inhibitors, other muscarinic receptor antagonistst (i.e., antichlolinergic agents), antiinfective agents (e.g. antibiotics or antivirals) and antihistamines.
  • anti-inflammatory agents e.g. steroidal anti-inflammatory agents, such as corticosteroids; and non-steroidal anti- inflammatory agents (NSATDs), phosphodiesterase IN inhibitors, other muscarinic receptor antagonistst (i.e., antichlolinergic agents), antiinfective agents (e.g. antibiotics or antivirals) and antihistamines.
  • NSATDs non-steroidal anti-inflammatory agents
  • Representative ⁇ 2 adrenergic receptor agonists that can be used in combination with, and in addition to, the compounds of this invention include, but are not limited to, salmeterol, salbutamol, formoterol, salmefamol, fenoterol, terbutaline, albuterol, isoetharine, metaproterenol, bitolterol, pirbuterol, levalbuterol and the like, or pharmaceutically acceptable salts thereof.
  • ⁇ 2 adrenergic receptor agonists that can be used in combination with the compounds of this invention include, but are not limited to, 3-(4- ⁇ [6-( ⁇ (2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl ⁇ amino)- hexyl]oxy ⁇ butyl)benzenesulfonamide and 3-(-3- ⁇ [7-( ⁇ (2R)-2-hydroxy-2-[4-hydroxy-3- (hydroxymethyl)phenyl] ethyl ⁇ -amino)heptyl] oxy ⁇ -propyl)benzenesulfonamide and related compounds disclosed in WO 02/066422, published on August 29, 2002; 3-[3-(4- ⁇ [6-([(2R)-2-hydroxy-2- [4-hydroxy-3 -(hydroxymethyl)phenyl] ethyl ⁇ amino)hexyl] - oxy ⁇ butyl)-phenyl]imidazol
  • Patent No. 6,576,793 Bl issued on June 10, 2003; N- ⁇ 2-[4-(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl ⁇ - (R)-2-hydroxy-2-(8-hydroxy-2(lH)-quinolinon-5-yl)ethylamine and related compounds disclosed in U.S. Patent No. 6,653,323 B2, issued on November 25, 2003; and pharmaceutically acceptable salts thereof.
  • the ⁇ 2 -adrenoreceptor agonist will be present in the pharmaceutical composition in a therapeutically effective amount.
  • the ⁇ -adrenoreceptor agonist will be present in an amount sufficient to provide from about 0.05 ⁇ g to about 500 ⁇ g per dose.
  • Representative steriodal anti-inflammatory agents that can be used in combination with the compounds of this invention include, but are not limited to, methyl prednisolone, prednisolone, dexamethasone, fluticasone propionate, 6,9-difluoro-17 -[(2- furanylcarbonyl)oxy]-l l-hydroxy-16-methyl-3-oxoandrosta-l,4-diene-17-carbothioic acid S-fluoromethyl ester, 6,9-difluoro-ll-hydroxy-16 -methyl-3-oxo-17 -propionyloxy- androsta-l,4-diene-17-carbothioic acid S-(2-oxotetrahydrofuran-3S-yl) ester, beclomethasone esters (e.g.
  • the steroidal anti-inflammatory agent is 6 ⁇ ,9 ⁇ -difluoro-17 ⁇ -[(2-furanylcarbonyl)oxy]-l l ⁇ -hydroxy- 16 ⁇ -methyl-3-oxoandrosta-l,4-diene-17 ⁇ -carbothioic acid S-fluoromethyl ester or a pharmaceutically acceptable salt or solvate thereof.
  • the steriodal anti- inflammatory agent will be present in the pharmaceutical composition in a therapeutically effective amount.
  • the steroidal anti-inflammatory agent will be present in an amount sufficient to provide from about 0.05 ⁇ g to about 500 ⁇ g per dose.
  • NSAIDs such as sodium cromoglycate; nedocromil sodium; phosphodiesterase (PDE) inhibitors (e.g. theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors); leukotriene antagonists (e.g. monteleukast); inhibitors of leukotriene synthesis; iNOS inhibitors; protease inhibitors, such as tryptase and elastase inhibitors; beta-2 integrin antagonists and adenosine receptor agonists or antagonists (e.g. adenosine 2a agonists); cytokine antagonists (e.g.
  • NSAIDs such as sodium cromoglycate; nedocromil sodium; phosphodiesterase (PDE) inhibitors (e.g. theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors)
  • leukotriene antagonists e.g. monteleukast
  • chemokine antagonists such as, an interleukin antibody (IL antibody), specifically, an IL-4 therapy, an E -13 therapy, or a combination thereof); or inhibitors of cytokine synthesis.
  • IL antibody interleukin antibody
  • PDE4 inhibitors or mixed PDE3/PDE4 inhibitors that can be used in combination with the compounds of this invention include, but are not limited to cis 4-cyano-4-(3-cyclopentyloxy-4- methoxyphenyl)cyclohexan-l-carboxylic acid, 2-carbomethoxy-4-cyano-4-(3- cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan- 1 -one; cis- [4-cyano-4-(3 - cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-l-ol]; cis-4-cyano-4-[3- (cyclopentyloxy)-4
  • PDE4 or mixed PDE4 PDE3 inhibitors include AWD-12-281 (elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough); CI-1018 or PD-168787 (Pfizer); benzodioxole compounds disclosed in WO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-l 1294A (Napp); roflumilast (Byk-Gulden); pthalazinone compounds disclosed in WO99/47505 (Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofylline (Almirall- Prodesfarma); VM554/UM565 (Nernalis); T-440 (Tanabe Seiyaku); and T2585 (Tanabe Seiyaku).
  • muscarinic antagonists i.e., anticholinergic agents
  • muscarinic antagonists include, but are not limited to, atropine, atropine sulfate, atropine oxide, methylatropine nitrate, homatropine hydrobromide, hyoscyamine (d, I) hydrobromide, scopolamine hydrobromide, ipratropium bromide, oxitropium bromide, tiotropium bromide, methantheline, propantheline bromide, anisotropine methyl bromide, clidinium bromide, copyrrolate (Robinul), isopropamide iodide, mepenzolate bromide, tridihexethyl chloride (Pathilone), hexocyclium methylsulfate, cyclopentolate hydrochloride, tropicamide, trihexyphenidyl hydrochloride,
  • antihistamines i.e., Hi-receptor antagonists
  • ethanolamines such as carbinoxamine maleate, clemastine fumarate, diphenylhydramine hydrochloride and dimenhydrinate
  • ethylenediamines such as pyrilamine amleate, tripelennamine hydrochloride and tripelennamine citrate
  • alkylamines such as chlorpheniramine and acrivastine
  • piperazines such as hydroxyzine hydrochloride, hydroxyzine pamoate, cyclizine hydrochloride, cyclizine lactate, meclizine hydrochloride and cetirizine hydrochloride
  • piperidines such as astemizole, levocabastine hydrochloride, loratadine or its descarboethoxy analogue, terfenadine and fexofenadine hydrochloride
  • ethanolamines such as carbinoxamine maleate, clemastine fumarate
  • Formulation Example A A dry powder for administration by inhalation is prepared as follows: Ingredients Amount
  • Salt of the invention 0.2 mg Lactose 25 mg ' Representative Procedure: The compound of the invention is micronized and then blended with lactose. This blended mixture is then loaded into a gelatin inhalation cartridge. The contents of the cartridge are administered using a powder inhaler.
  • Formulation Example B A dry powder formulation for use in a dry powder inhalation device is prepared as follows: Representative Procedure: A pharmaceutical composition is prepared having a bulk formulation ratio of micronized salt of the invention to lactose of 1 :200. The composition is packed into a dry powder inhalation device capable of delivering between about 10 ⁇ g and about 100 ⁇ g of the compound of the invention per dose.
  • a dry powder for administration by inhalation in a metered dose inhaler is prepared as follows: Representative Procedure: A suspension containing 5 wt. % of a salt of the invention and 0.1 wt. % lecithin is prepared by dispersing 10 g of the compound of the invention as micronized particles with mean size less than 10 ⁇ m in a solution formed from 0.2 g of lecithin dissolved in 200 mL of demineralized water. The suspension is spray dried and the resulting material is micronized to particles having a mean diameter less than 1.5 ⁇ m. The particles are loaded into cartridges with pressurized 1,1,1,2- tetrafluoroethane.
  • a pharmaceutical composition for use in a metered dose inhaler is prepared as follows: Representative Procedure: A suspension containing 5 % salt of the invention, 0.5 % lecithin, and 0.5 % trehalose is prepared by dispersing 5 g of active ingredient as micronized particles with mean size less than 10 m in a colloidal solution formed from 0.5 g of trehalose and 0.5 g of lecithin dissolved in 100 mL of demineralized water. The suspension is spray dried and the resulting material is micronized to particles having a mean diameter less than 1.5 ⁇ m. The particles are loaded into canisters with pressurized 1,1,1 ,2-tetrafluoroethane.
  • a pharmaceutical composition for use in a nebulizer inhaler is prepared as follows: Representative Procedure: An aqueous aerosol formulation for use in a nebulizer is prepared by dissolving 0.1 mg of the salt of the invention in 1 mL of a 0.9 % sodium chloride solution acidified with citric acid. The mixture is stirred and sonicated until the active ingredient is dissolved. The pH of the solution is adjusted to a value in the range of from 3 to 8 by the slow addition of NaOH.
  • Hard gelatin capsules for oral administration are prepared as follows: Ingredients Amount
  • a suspension for oral administration is prepared as follows: Ingredients Amount Salt of the invention 1.0 g Fumaric acid 0.5 g Sodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 g Granulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Neegum k (Nanderbilt Co.) 1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilled water q.s. to lOO mL
  • Salt of the invention 0.2 g Sodium acetate buffer solution (0.4 M) 2.0 mL HC1 (0.5 ⁇ ) or ⁇ aOH (0.5 ⁇ ) q.s. to pH 4 Water (distilled, sterile) q.s. to 20 mL
  • Such medical conditions include, by way of example, pulmonary disorders or diseases associated with reversible airway obstruction, such as chronic obstructive pulmonary disease (e.g., chronic and whez bronchitis and emphysema), asthma, pulmonary fibrosis and the like.
  • chronic obstructive pulmonary disease e.g., chronic and whez bronchitis and emphysema
  • asthma pulmonary fibrosis
  • Other conditions which are expected to be treated include premature labor, depression, congestive heart failure, skin diseases (e.g., inflammatory, allergic, psoriatic and proliferative skin diseases, conditions where lowering peptic acidity is desirable (e.g., peptic and gastric ulceration) and muscle wasting disease.
  • skin diseases e.g., inflammatory, allergic, psoriatic and proliferative skin diseases, conditions where lowering peptic acidity is desirable (e.g., peptic and gastric ulceration) and muscle
  • this invention is directed to a method for treating a pulmonary disorder, the method comprising administering to a patient in need of treatment a therapeutically effective amount of pharmaceutical composition prepared from a crystalline salt of this invention, the pharmaceutical composition comprising a naphthalene-l,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy- 2-(8-hydroxy-2-oxo- 1 ,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester or a solvate thereof.
  • a pharmaceutical composition prepared from a crystalline salt may or may not contain the salt in crystalline form.
  • a salt of this invention When used to treat a pulmonary disorder, a salt of this invention will typically be administered by inhalation in multiple doses per day, in a single daily dose or a single weekly dose. Generally, the dose for treating a pulmonary disorder will range from about 10 ⁇ g/day to about 200 ⁇ g/day. When administered by inhalation, the compounds of this invention typically have the effect of producing bronchodilation.
  • this invention is directed to a method of producing bronchodilation in a patient, the method comprising administering to a patient a bronchodilation-producing amount of the pharmaceutical composition.
  • the dose for producing brochodilation will range from about 10 ⁇ g/day to about 200 ⁇ g/day.
  • this invention is directed to a method of treating chronic obstructive pulmonary disease or asthma, the method comprising administering to a patient in need of treatment a therapeutically effective amount of the pharmaceutical composition.
  • a salt of this invention will typically be administered by inhalation in multiple doses per day or in a single daily dose.
  • the dose for treating COPD or asthma will range from about 10 ⁇ g/day to about 200 ⁇ g/day.
  • COPD includes chronic obstructive bronchitis and emphysema (see, for example, Barnes, Chronic Obstructive Pulmonary Disease, NEngl J Med 2000: 343:269-78).
  • a salt of this invention is optionally administered in combination with other therapeutic agents.
  • the pharmaceutical compositions and methods of this invention further comprise a therapeutically effective amount of a steroidal anti-inflammatory agent.
  • naphthalene-l,5-disulfonic acid salt of this invention can be demonstrated using various in vitro and in vivo assays well-known to those skilled in the art. For example, representative assays are described in further detail in the following Examples.
  • HPLC analysis was conducted using an Agilent (Palo Alto, CA) Series 1100 instrument with Zorbax Bonus RP 2.1 x 50 mm columns, supplied by Agilent, (a C14 column), having a 3.5 micron particle size. Detection was by UN absorbance at 214 nm. HPLC 10-70 data was obtained with a flow rate of 0.5 mL/minute of 10%-70% B over 6 minutes.
  • Mobile phase A was 2 % - 98 % - 0.1% ACN-H 2 O-TFA; and mobile phase B was 90 % - 10 % - 0.1 % ACN-H 2 O-TFA.
  • HPLC 5-35 data and HPLC 10-90 data were obtained with a 5 minute gradient.
  • Liquid chromatography mass spectrometry (LCMS) data were obtained with an Applied Biosystems (Foster City, CA) model API-150EX instrument.
  • LCMS 10-90 data was obtained with a 10 % - 90 % mobile phase B over a 5 minute gradient.
  • Small scale purification was conducted using an API 150EX Prep Workstation system from Applied Biosystems.
  • the mobile phase was A: water + 0.05% v/v TFA; and B: acetonitrile + 0.05% v/v TFA.
  • arrays typically about 3 to 50 mg recovered sample size
  • the following conditions were used: 20 mL/min flow rate; 15 min gradients and a 20 mm x 50 mm Prism RP column with 5 micron particles (Thermo Hypersil-Keystone, Bellefonte, PA).
  • For larger scale purifications typically greater than 100 mg crude sample, the following conditions were used: 60 mL/min flow rate; 30 min gradients and a 41.4 mm x 250 mm Microsorb BDS column with 10 micron particles (Varian, Palo Alto, CA).
  • Preparation 4 8-BenzyIoxy-5-(2-Bromoacetyl)-lH-quinolin-2-one
  • the product of Preparation 3 (20.0 g, 68.2 mmol) was dissolved in dichloromethane (200 mL) and cooled to 0°C.
  • Boron trifluoride diethyl etherate (10.4 mL, 82.0 mmol) was added via syringe and the mixture was warmed to room temperature to give a thick suspension.
  • the suspension was heated at 45 °C (oil bath) and a solution of bromine (11.5 g, 72.0 mmol) in dichloromethane (100 mL) was added over 40 min.
  • the mixture was kept at 45 °C for an additional 15 min and then cooled to room temperature.
  • the mixture was concentrated under reduced pressure and then triturated with 10% aqueous sodium carbonate (200 mL) for 1 hour.
  • the solids were collected on a Buchner funnel, washed with water (4 x 100 mL) and dried under reduced pressure.
  • the product of two runs was combined for purification.
  • the crude product (52 g) was triturated with 50% methanol in chloroform (500 mL) for 1 hour.
  • the product was collected on a Buchner funnel and washed with 50% methanol in chloroform (2 x 50 mL) and methanol (2 x 50 mL).
  • the solid was dried under reduced pressure to give the title compound (34.1 g) as a powder.
  • Preparation 6 8-Benzyloxy-5-[(R)-2-bromo-l-(tert-butyldimethylsiIanyloxy)ethyI]-lH-quinoIin-2- one
  • NN- dimethylformamide 260 mL
  • 2,6-Lutidine 40.3 g, 376 mmol
  • tert-butyldimethylsilyl trifluoromethanesulfonate 99.8 g, 378 mmol
  • Biphenyl-2-ylcarbamic Acid Piperidin-4-yI Ester Biphenyl-2-isocyanate (97.5 g, 521 mmol) and 4-hydroxy-l-benzylpiperidine (105 g, 549 mmol), both commercially-available from Aldrich, Milwaukee, WI, were heated together at 70 °C for 12 h, during which time the formation of biphenyl-2-ylcarbamic acid l-benzylpiperidin-4-yl ester was monitored by LCMS. The reaction mixture was then cooled to 50 °C and ethanol (1 L) was added, and then 6M hydrochloric acid (191 mL) was added slowly.
  • reaction mixture was then cooled to ambient temperature and ammonium formate (98.5 g, 1.56 mol) was added and nitrogen gas was bubbled through the solution vigorously for 20 min. Palladium (10 wt. % (dry basis) on activated carbon) (20 g) was then added. The reaction mixture was heated at 40 °C for 12 h and then filtered through a pad of Celite. The solvent was then removed under reduced pressure and 1M hydrochloric acid (40 mL) was added to the crude residue. Sodium hydroxide (ION) was then added to adjust the pH to 12.
  • reaction mixture was then cooled back down to 0 °C and a solution of 1,9-dibromononane (2.46 mL, 12.1 mmol) in dimethylformamide (100 mL) was added.
  • the reaction mixture was stirred overnight at room temperature. After 24 h, MS analysis showed that the reaction was completed.
  • the reaction mixture was concentrated to dryness and diluted with ethyl acetate (100 mL). The organic layer was washed with saturated sodium bicarbonate (2 x 100 mL), brine
  • Triethylamine hydrogen fluoride (376 ⁇ L, 2.3 mmol) was added to a solution of the product of Preparation 11 (1.3 g, 1.5 mmol) in THF (8 mL) and the reaction mixture was stirred at ambient temperature. After 5 h, the reaction was complete as determined by LCMS analysis.
  • reaction mixture was then quenched with IN NaOH until the pH was 14 and then diluted with ethyl acetate (20 mL) and washed with IN NaOH (20 mL) and brine (20 mL). The organic phase was then separated, dried over magnesium sulfate, and concentrated to yield the title compound (1.1 g).
  • a solution of the product of Preparation 12 (1.1 g, 1.5 mmol) was flushed with nitrogen and palladium on carbon (10%, 110 mg) was added. The reaction mixture was stirred under hydrogen at balloon pressure. Analysis by LCMS showed that the reaction was completed after 9 h. The reaction mixture was then filtered and concentrated to yield a yellow solid.
  • TEMPO 2,2,6,6-Tetramethyl-l-piperidinyloxy free radical
  • Biphenyl-2-ylcarbamic Acid l-(9,9-Dimethoxynony ⁇ )piperidin-4-yl Ester To a 50 mL three-necked, round-bottomed flask was added biphenyl-2-ylcarbamic acid piperidin-4-yl ester (1 g, 3.38 mmol) and acetonitrile (10 mL) to form a slurry.
  • Biphenyl-2-yIcarbamic Acid l-(9-Oxononyl)piperidin-4-yI Ester To a 500 mL round-bottomed flask with a magnetic stirrer was added biphenyl-2- ylcarbamic acid l-(9,9-dimethoxynonyl)piperidin-4-yl ester (7.7 g, 15.9 mmol) and then acetonitrile (70 mL) and aqueous 1M hydrochloric acid (70 mL). The resulting mixture was stirred at room temperature for 1 h and then dichloromethane (200 mL) was added. This mixture was stirred for 15 min. and then the layers were separated. The organic layer was dried (MgSO 4 ), filtered and concentrated under reduce pressure to afford the title intermediate (6.8 g), which was used in the next step without further purification.
  • the residue was purified by silica gel chromatography (15g silica/1 g crude) using 5% MeOH in DCM/0.5% NH 4 OH (10 x 150 mL), 8% MeOH in DCM/0.5% NH 4 OH (10 x 150 mL) and 10% MeOH in DCM/0.5% NH 4 OH (10 x 150 mL). The appropriate fractions were combined and the solvent was removed under reduced pressure while maintaining the temperature ⁇ 35 °C to give the title intermediate (4.05 g , 97% purity).
  • the resulting mixture was flushed with nitrogen and then stirred at 90 °C for 6 hours.
  • the mixture was then cooled to room temperature and water (300 mL) and ethyl acetate (300 mL) were added.
  • the layers were separated and the organic layer was washed with water (200 mL), a 1:1 mixture of water and aqueous saturated sodium chloride solution (200 mL), and water (200 mL).
  • the organic layer was then dried over magnesium sulfate, filtered and concentrated under reduced pressure to provide the title compound as an orange oil.
  • To the orange oil was added heptane (200 mL) and ethyl acetate (200 mL) and the resulting mixture was heated to 65 °C to produce a clear solution.
  • biphenyl-2-ylcarbamic acid l-(9,9- dimethoxynonyl)piperidin-4-yl ester (20 g, 41.8 mmol) and acetonitrile 50 mL).
  • the flask was then purged with hydrogen gas for 5 minutes and then palladium hydroxide (3.5 g, 20% Pd (dry basis) on carbon, 60% water) was added.
  • the resulting mixture was stirred at room temperature while hydrogen gas was slowly bubbled through the mixture for about 5 hours (>98% conversion by HPLC).
  • the resulting crystalline solid was then collected by filtration, washed with methanol (20 mL) and air dried to give the title compound as an off-white crystalline solid (8 g).
  • the crystalline solid was then re-slurried in a 20% mixture of methanol in water (80 mL total) and stirred for 24 hours.
  • the crystalline solid was then collected by filtration and dried to give the title compound as a crystalline solid (7.1 g, >98% purity by HPLC).
  • X-Ray Powder Diffraction X-Ray powder diffraction patterns were obtained with a Shimadzu 6000 diffractometer using Cu K (40.0 kN, 35.0 mA) radiation. The analysis was typically performed with the goniometer running in continuous-scan mode of 2 °/min with a step size of 0.02 ° over a range of 4 to 45 ° in two-theta angle. Samples were prepared on glass specimen holders as a thin layer of powdered material. The instrument was calibrated to a silicon metal standard. A smoothing and background subtraction process was performed on the raw spectrum according to standard procedures.
  • DSC Differential scanning calorimetry
  • Theraiogravimetric analysis (TGA) was performed using a TA Instruments Model Q-50 module equipped with Hi-Resolution capability. Data were collected and analyzed using TA Instruments Thermal Solutions software.
  • a sample weighing about 10 mg was placed onto a platinum pan and scanned with a high resolution-heating rate from ambient temperature to 300 °C. The balance and furnace chambers were purged with nitrogen flows during use.
  • a representative TGA trace for a sample of crystalline naphthalene- 1,5- disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2-hydroxy-2-(8-hydroxy-2- oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ -piperidin-4-yl ester (prepared as described in Example 3) is shown in Figure 2.
  • the DSC trace demonstrates that a naphthalene- 1,5-disulfonic acid salt of the present invention has excellent thermal stability with the melting peak at about 220 °C and no thermal decomposition below 200 °C.
  • the DSC trace shows an onset of endothermic heat flow at about 200 °C.
  • the TGA trace indicates that a naphthalene- 1,5- disulfonic acid salt of the present invention lost a small amount (about 1.8%) in weight from room temperature to 150 °C, which is consistent with the loss of residual moisture or solvent.
  • Dynamic Moisture Sorption Gravimetry (DMSG or DMS) assessment (also known as a moisture sorption-desorption profile) was determined for a sample of a crystalline naphthalene- 1,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l- ⁇ 9-[(R)-2- hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro-quinolin-5-yl)ethylamino]nonyl ⁇ piperidin-4-yl ester (prepared as described in Example 3) using a NTI atmospheric microbalance, SGA- 100 system. Approximately 10 mg of sample was used in the microbalance and the sample was run as received.
  • DMSG or DMS Dynamic Moisture Sorption Gravimetry
  • the humidity was set at ambient condition on the day the analysis began.
  • a typical DMSG analysis consisted of three scans: ambient to 2% relative humidity (RH), 2% RH to 90% RH, 90% RH to 5% RH.
  • the scan rate was 5 % RH/step.
  • the mass was measured every two minutes using an equilibrium window of 5 points within 0.01% of the initial sample weight.
  • the RH was changed to the next value (+/- 5%RH) when the mass of the sample was stable to within 0.01%) for 5 consecutive points.
  • a representative DMSG trace is shown in Figure 3.
  • the DMS trace demonstrates that a naphthalene-l,5-disulfonic acid salt of the present invention has a reversible sorption/desorption profile with moderate ( ⁇ 5%) hygroscopicity.
  • the salt has an acceptable 3.7% weight gain when it exposed to a broad humidity range from 2% RH up to 90% RH and it has a less than 1.5% weight gain in the humidity range of 40% RH to 75% RH.
  • the reversible moisture sorption/desorption profile demonstrates that the crystalline salt of the present invention possesses an acceptable hygroscopicity and is not deliquescent.
  • the infrared (IR) absorption spectrum was determined over the wave number ( ⁇ ) range 4000 to 675 cm “1 using an Avatar 360 FT-IR spectrometer equipped with a Nicolet omnis sample attenuated total reflection (ATR) sample holder.
  • formate buffer 2 % acetonitrile
  • Mobile Phase B 10% formate buffer, 90 % acetonitrile
  • formate buffer 10 mM ammonium formate buffer, pH 3.8 adjusted with formic acid
  • flow rate 1.2 mL/min
  • gradient 0 to 40 % B over 40 min, 40 to 100 % B over 25 min, 100 to 0 % B over 1 min; 0 % B for 7 min
  • detection wavelength 258 nm
  • injection volume 10 ⁇ L
  • sample concentration 0.5 mg/mL in 1:1 MPA:MPB.
  • the initial purity of the samples was 99.4 % as determined by HPLC area percentage.
  • ⁇ i and ⁇ 2 receptor expressing membranes For preparation of ⁇ i and ⁇ 2 receptor expressing membranes, cell pellets were resuspended in lysis buffer (10 mM HEPES/HC1, lOmM EDTA, pH 7.4 at 4°C) and homogenized using a tight-fitting Dounce glass homogenizer (30 strokes) on ice.
  • lysis buffer 10 mM HEPES/HC1, lOmM EDTA, pH 7.4 at 4°C
  • cell pellets were homogenated in lysis buffer (lOmM Tris/HCl, pH 7.4) supplemented with one tablet of "Complete Protease Inhibitor Cocktail Tablets with 2 mM EDTA" per 50 mL buffer (Roche Catalog No. 1697498, Roche Molecular Biochemicals, Indianapolis, IN).
  • a Radioligand Binding Assay for Human ⁇ i, ⁇ 2 and ⁇ 3 Adrenergic Receptors Binding assays were performed in 96-well microtiter plates in a total assay volume of 100 ⁇ L with 10-15 ⁇ g of membrane protein containing the human ⁇ i, ⁇ 2 or ⁇ 3 adrenergic receptors in assay buffer (75 mM Tris/HCl pH 7.4 at 25°C, 12.5 mM MgCl 2 , 1 mM EDTA, 0.2% BSA). Saturation binding studies for determination of K ⁇ values of the radioligand were done using [ 3 H]-dihydroalprenolol (NET-720, 100 Ci/mmol,
  • Non-specific binding was determined in the presence of 10 ⁇ M propranolol. Assays were incubated for 1 hour at 37°C, and then binding reactions were terminated by rapid filtration over GF/B for the ⁇ i and ⁇ 2 receptors or GF/C glass fiber filter plates for the ⁇ 3 receptors (Packard BioScience Co., Meriden, CT) presoaked in 0.3% polyethyleneimine. Filter plates were washed three times with filtration buffer (75 mM Tris/HCl pH 7.4 at 4°C, 12.5 mM MgCl 2 , 1 mM EDTA) to remove unbound radioactivity.
  • filtration buffer 75 mM Tris/HCl pH 7.4 at 4°C, 12.5 mM MgCl 2 , 1 mM EDTA
  • K ; - values for test compounds were calculated from observed IC 5 Q values and the K d value of the radioligand using the Cheng-Prusoff equation (Cheng Y, and Prusoff WH., Biochemical Pharmacology, 1973, 22, 23, 3099-108). In this assay, a lower K, value indicates that a test compound has a higher binding affinity for the receptor tested.
  • the compound of formula I was found to have a K ; - value of less than about 10 nM for the ⁇ 2 adrenergic receptor.
  • Radioligand Binding Assay for Muscarinic Receptors Radioligand binding assays for cloned human muscarinic receptors were performed in 96-well microtiter plates in a total assay volume of 100 ⁇ L. CHO cell membranes stably expressing either the hMi, hM 2 , hM 3 , hM 4 or hM 5 muscarinic subtype were diluted in assay buffer to the following specific target protein concentrations
  • test compounds were initially dissolved to a concentration of 400 ⁇ M in dilution buffer and then serially diluted 5x with dilution buffer to final concentrations ranging from 10 pM to 100 ⁇ M.
  • the addition order and volumes to the assay plates were as follows: 25 ⁇ L radioligand, 25 ⁇ L diluted test compound, and 50 ⁇ L membranes. Assay plates were incubated for 60 minutes at 37°C.
  • Binding reactions were terminated by rapid filtration over GF/B glass fiber filter plates (PerkinElmer Inc., Wellesley, MA) pre-treated in 1% BSA. Filter plates were rinsed three times with wash buffer (10 mM HEPES) to remove unbound radioactivity. The plates were then air dried and 50 ⁇ L Microscint-20 liquid scintillation fluid (PerkinElmer hie, Wellesley, MA) was added to each well. The plates were then counted in a PerkinElmer Topcount liquid scintillation counter (PerkinElmer Inc., Wellesley, MA).
  • Binding data were analyzed by nonlinear regression analysis with the GraphPad Prism Software package (GraphPad Software, Inc., San Diego, CA) using the one-site competition model.
  • K values for test compoimds were calculated from observed IC 50 values and the KQ value of the radioligand using the Cheng-Prusoff equation (Cheng Y; Prusoff WH. (1973) Biochemical Pharmacology, 22(23):3099-108).
  • Kj values were converted to pKi values to determine the geometric mean and 95% confidence intervals. These summary statistics were then converted back to Kj values for data reporting. In this assay, a lower K; value indicates that the test compound has a higher binding affinity for the receptor tested.
  • the compound of formula I was found to have a K; value of less than about 10 nM for the M 3 muscarinic receptor.
  • CHO-K1 cell lines stably expressing cloned human ⁇ i, ⁇ 2 or ⁇ 3 adrenergic receptors were grown to near confluency in HAM's F-12 media supplemented with 10% FBS and Geneticin (250 ⁇ g/mL). Cells were rinsed with PBS and detached in dPBS (Dulbecco's Phosphate Buffered Saline, without CaCl 2 and MgCl 2 ) containing 2 mM EDTA or Trypsin-EDTA solution (0.05% trypsin/0.53 mM EDTA).
  • dPBS Dynabecco's Phosphate Buffered Saline, without CaCl 2 and MgCl 2
  • Trypsin-EDTA solution 0.05% trypsin/0.53 mM EDTA
  • test compounds (10 mM in DMSO) were diluted into PBS containing 0.1% BSA in Beckman Biomek-2000 and tested at 11 different concentrations ranging from 100 ⁇ M to 1 pM.
  • Cells are rinsed once with dPBS and lifted with Trypsin-EDTA solution (0.05% trypsin/0.53 mM EDTA) as described in the Cell Culture and Membrane Preparation section above.
  • Trypsin-EDTA solution 0.05% trypsin/0.53 mM EDTA
  • the detached cells are washed twice by centrifugation at 650 x g for five minutes in 50 mL dPBS.
  • the cell pellet is then re-suspended in 10 mL dPBS, and the cells are counted with a Coulter Zl Dual Particle Counter (Beckman Coulter, Fullerton, CA).
  • the cells are centrifuged again at 650 x g for five minutes and re-suspended in stimulation buffer to an assay concentration of 1.6 x 10 6 - 2.8 x 10 6 cells/mL.
  • the test compound is initially dissolved to a concentration of 400 ⁇ M in dilution buffer (dPBS supplemented with 1 mg/mL BSA (0.1%)), and then serially diluted with dilution buffer to final molar concentrations ranging from 100 ⁇ M to 0.1 nM.
  • Oxotremorine is diluted in a similar manner.
  • oxotremorine inhibition of adenylyl cyclase (AC) activity 25 ⁇ L forskolin (25 ⁇ M final concentration diluted in dPBS), 25 ⁇ L diluted oxotremorine, and 50 ⁇ L cells are added to agonist assay wells.
  • 25 ⁇ L forskolin and oxotremorine 25 ⁇ M and 5 ⁇ M final concentrations, respectively, diluted in dPBS
  • 25 ⁇ L diluted test compound, and 50 ⁇ L cells are added to remaining assay wells.
  • the Cheng- Prusoff equation is used to calculate the Kj, using the EC 50 of the oxotremorine concentration-response curve and the oxotremorine assay concentration as the K D and [L], respectively.
  • a lower K; value indicates that the test compound has a higher functional activity at the receptor tested.
  • the compound of formula I was found to have a K; value of less than about 10 nM for blockade of oxotremorine-inhibition of forskolin- mediated cAMP accumulation in CHO-K1 cells expressing the hM receptor.
  • the functional potency of test compounds can be determined by measuring the ability of the compounds to block oxotremorine-stimulated [ 35 S]GTP ⁇ S_binding in CHO-K1 cells expressing the hM 2 receptor.
  • frozen membranes were thawed and then diluted in assay buffer with a final target tissue concentration of 5-10 ⁇ g protein per well. The membranes were briefly homogenized using a Polytron PT-2100 tissue disrupter and then added to the assay plates.
  • the EC 90 value (effective concentration for 90% maximal response) for stimulation of [ 35 S]GTP ⁇ S binding by the agonist oxotremorine was determined in each experiment. To determine the ability of a test compound to inhibit oxotremorine-stimulated
  • [ 35 S]GTP ⁇ S binding the following was added to each well of 96 well plates: 25 ⁇ L of assay buffer with [ 35 S]GTP ⁇ S (0.4nM), 25 ⁇ L of oxotremorme(EC 90 ) and GDP (3uM), 25 ⁇ L of diluted test compound and 25 ⁇ L CHO cell membranes expressing the hM 2 receptor.
  • the assay plates were then incubated at 37 °C for 60 minutes.
  • the assay plates were filtered over 1% BSA-pretreated GF/B filters using a PerkinElmer 96-well harvester. The plates were rinsed with ice-cold wash buffer for 3 x 3 seconds and then air or vacuum dried.
  • Microscint-20 scintillation liquid 50 ⁇ L was added to each well, and each plate was sealed and radioactivity counted on a Topcounter (PerkinElmer). Data were analyzed by nonlinear regression analysis with the GraphPad Prism Software package (GraphPad Software, Inc., San Diego, CA) using the non-linear regression, one-site competition equation. The Cheng-Prusoff equation was used to calculate the K;, using the IC 50 values of the concentration-response curve for the test compound and the oxotremorine concentration in the assay as the K D and [L], ligand concentration, respectively. In this assay, a lower Kj value indicates that the test compound has a higher functional activity at the receptor tested.
  • the compound of formula I was found to have a Kj value of less than about 10 nM for blockade of oxotremorine-stimulated [ 35 S]GTP ⁇ S_ binding in CHO-K1 cells expressing the hM 2 receptor.
  • the FLIPR (Molecular Devices, Sunnyvale, CA) assay capitalizes on this increase in intracellular calcium by using a calcium sensitive dye (Fluo-4AM, Molecular Probes, Eugene, OR) that fluoresces when free calcium binds. This fluorescence event is measured in real time by the FLIP R, which detects the change in fluorescence from a monolayer of cells cloned with human Mi and M 3 , and chimpanzee M 5 receptors.
  • Antagonist potency can be determined by the ability of antagonists to inhibit agonist- mediated increases in intracellular calcium.
  • CHO cells stably expressing the hMi, hM 3 and cM 5 receptors are seeded into 96-well FLIPR plates the night before the assay is done. Seeded cells are washed twice by Cellwash (MTX Labsystems, Inc.) with FLIPR buffer (10 mM HEPES, pH 7.4, 2 mM calcium chloride, 2.5 mM probenecid in Hank's Buffered Salt Solution (HBSS) without calcium and magnesium) to remove growth media and leaving 50 ⁇ L/well of FLIPR buffer.
  • FLIPR buffer 10 mM HEPES, pH 7.4, 2 mM calcium chloride, 2.5 mM probenecid in Hank's Buffered Salt Solution (HBSS) without calcium and magnesium
  • the cells are then incubated with 50 ⁇ L/well of 4 ⁇ M FLUO-4AM (a 2X solution was made) for 40 minutes at 37 °C, 5% carbon dioxide. Following the dye incubation period, cells are washed two times with FLIPR buffer, leaving a final volume of 50 ⁇ L/well.
  • FLUO-4AM a 2X solution was made
  • the dose-dependent stimulation of intracellular Ca 2+ release for oxotremorine is first determined so that antagonist potency can later be measured against oxotremorine stimulation at an EC 90 concentration.
  • Cells are first incubated with compound dilution buffer for 20 minutes, followed by agonist addition, which is performed by the FLIPR.
  • An oxotremorine concentration of 3 x EC F is prepared in stimulation plates such that an EC 90 concentration of oxotremorine is added to each well in the antagonist inhibition assay plates.
  • the parameters used for the FLIPR are: exposure length of 0.4 seconds, laser strength of 0.5 watts, excitation wavelength of 488 nm, and emission wavelength of 550 nm. Baseline is determined by measuring the change in fluorescence for 10 seconds prior to addition of agonist.
  • the FLIPR continuously measured the change of fluorescence every 0.5 to 1 second for 1.5 minutes to capture the maximum fluorescence change.
  • the change of fluorescence is expressed as maximum fluorescence minus baseline fluorescence for each well.
  • the raw data is analyzed against the logarithm of drug concentration by nonlinear regression with GraphPad Prism (GraphPad Software, Inc., San Diego, CA) using the built-in model for sigmoidal dose-response.
  • Antagonist Kj values are determined by Prism using the oxotremorine EC 50 value as the K D and the oxotremorine EC 0 for the ligand concentration according to the Cheng-Prusoff equation (Cheng & Prusoff, 1973).
  • a lower Kj value indicates that the test compound has a higher functional activity at the receptor tested.
  • the compound of formula I was found to have a Kj value of less than about 10 nM for blockade of agonist-mediated calcium release in CHO cells stably expressing the hMi, hM 3 and cM 5 receptors.
  • ⁇ 2 Adrenergic Receptor For the determination of agonist potencies and efficacies (intrinsic activities) in a cell line expressing endogenous levels of the ⁇ 2 adrenergic receptor, a human lung epithelial cell line (BEAS-2B) was used (ATCC CRL-9609, American Type Culture Collection, Manassas, NA) (January B, et al., British Journal of Pharmacology, 1998, 123, 4, 701-11). Cells were grown to 75-90% confluency in complete, serum-free medium (LHC-9 MEDIUM containing Epinephrine and Retinoic Acid, cat # 181-500, Biosource International, Camarillo, CA).
  • Test compounds were serially diluted into assay buffer (75 mM Tris/HCl pH 7.4 at 25°C, 12.5 mM MgCl 2 , 1 mM EDTA, 0.2% BSA) in Beckman Biomek-2000. Test compoimds were tested in the assay at 11 different concentrations, ranging from 10 ⁇ M to 10 pM.
  • Reactions were incubated for 10 min at 37 °C and stopped by addition of 100 ⁇ L of ice-cold detection buffer. Plates were sealed, incubated over night at 4 °C and counted the next morning in a Topcount scintillation counter (Packard BioScience Co., Meriden, CT). The amount of cAMP produced per mL of reaction was calculated based on the counts observed for samples and cAMP standards, as described in the manufacturer's user manual. Data were analyzed by nonlinear regression analysis with the GraphPad Prism Software package (GrapliPad Software, Inc., San Diego, CA) using the 4-parameter model for sigmoidal dose-response.
  • a lower EC 50 value indicates that the test compound has a higher functional activity at the receptor tested.
  • the compound of formula I was found to have a EC 50 value of less than about 10 nM for the ⁇ 2 adrenergic receptor.
  • each guinea pig was anesthetized with an intramuscular injection of ketamine (43.75 mg/kg), xylazine (3.50 mg/kg) and acepromazine (1.05 mg/kg).
  • ketamine 43.75 mg/kg
  • xylazine 3.50 mg/kg
  • acepromazine 1.05 mg/kg
  • the surgical site was shaved and cleaned with 70% alcohol, a 2-3 cm midline incision of the ventral aspect of the neck was made.
  • the jugular vein was isolated and cannulated with a saline-filled polyethylene catheter (PE-50, Becton Dickinson, Sparks, MD) to allow for intravenous infusions of acetylcholine (Ach) or histamine in saline.
  • PE-50 saline-filled polyethylene catheter
  • the trachea was then dissected free and cannulated with a 14G teflon tube (#NE- 014, Small Parts, Miami Lakes, FL). If required, anesthesia was maintained by additional intramuscular injections of the aforementioned anesthetic mixture. The depth of anesthesia was monitored and adjusted if the animal responds to pinching of its paw or if the respiration rate was greater than 100 breaths/minute.
  • the animal was placed into a plethysmograph (#PLY3114, Buxco Electronics, hie, Sharon, CT) and an esophageal pressure camiula (PE-160, Becton Dickinson, Sparks, MD) was inserted to measure pulmonary driving pressure (pressure).
  • the teflon tracheal tube was attached to the opening of the plethysmograph to allow the guinea pig to breathe room air from outside the chamber.
  • the chamber was then sealed.
  • a heating lamp was used to maintain body temperature and the guinea pig's lungs were inflated 3 times with 4 mL of air using a
  • Ach (Sigma- Aldrich, St. Louis, MO) (0.1 mg/mL) was infused intravenously for 1 minute from a syringe pump (sp210iw, World Precision Instruments, Inc., Sarasota, FL) at the following doses and prescribed times from the start of the experiment: 1.9 ⁇ g/minute at 5 minutes, 3.8 ⁇ g/minute at 10 minutes, 7.5 ⁇ g/minute at 15 minutes, 15.0 ⁇ g/minute at 20 minutes, 30 ⁇ g/minute at 25 minutes and 60 ⁇ g/minute at 30 minutes.
  • bronchoprotection of test compounds was assessed in the acetylcholine challenge model without prefreatment with a beta blocking compound.
  • histamine 25 ⁇ g/mL (Sigma- Aldrich, St. Louis, MO) was infused intravenously for 1 minute from a syringe pump at the following doses and prescribed times from the start of the experiment: 0.5 ⁇ g/minute at 5 minutes, 0.9 ⁇ g/minute at 10 minutes,
  • pulmonary parameters include respiration frequency (breaths/minute), compliance (mL/cm H O) and pulmonary resistance (cm H O/ mL per second).
  • Inhibition dose-response curves for 'R L ' were fitted with a four parameter logistic equation using GraphPad Prism, version 3.00 for Windows (GraphPad Software, San Diego, California) to estimate bronchoprotective ID 5 o (dose required to inhibit the ACh (60 ⁇ g/min) bronchocontrictor response by 50%).
  • Ci concentration of Ach or histamine preceding C 2
  • C 2 concentration of Ach or histamine resulting in at least a 2-fold increase in pulmonary resistance
  • R ⁇ R Q Baseline
  • the compound of formula I was found to have an ID 50 less than about 100 ⁇ g/mL for ACh-induced bronchoconstriction and an ID 5 Q less than about 100 ⁇ g/mL for His- induced bronchoconstriction at 1.5 hours post-dose. Additionally, the compound of formula I was found to have a duration (PD T ⁇ / ) of brochoprotective activity of at least about 24 hours post-dose.

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Abstract

L'invention concerne un sel acide cristallin de naphthlène-1,5-bisulfonique d'ester 1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)éthylamino]nonyl}pipéridin-4-yl d'acide biphényl-2-ylcarbamique ou un solvate de celui-ci. L'invention concerne également des compositions pharmaceutiques comprenant un tel sel ou préparé au moyen d'un tel sel; des procédés et des intermédiaires pour préparer un tel sel, ainsi que des méthodes d'utilisation d'un tel sel pour traiter un trouble pulmonaire.
PCT/US2005/004392 2004-02-13 2005-02-11 Cristalline d'un compose biphenyle Ceased WO2005080375A1 (fr)

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